Project Summary/Abstract A significant percentage of HIV-positive individuals who start antiretroviral therapy (ART) with a low CD4+ T cell count have CD4 counts that plateau at abnormally low levels despite years of virologic suppression on ART. The risk of death for these suboptimal immune responders (SoIRs) is 2-3 times higher than that of individuals whose CD4 count rises appropriately with ART, and this higher risk of death persists for a decade or more. The mechanisms underlying the suboptimal immune recovery and increased mortality rates in SoIRs remain poorly defined. One clear association has emerged: SoIRs have significantly higher levels of immune activation than other ART-treated individuals. We know that stimulation of HIV-infected CD4s through the T cell receptor results in HIV RNA and protein production and recognition by HIV-specific cytotoxic T lymphocytes. This suggests that the usual daily antigenic stimulation of CD4s could produce excess immune activation in individuals with a very large burden of latent HIV. Concordantly, a correlation between the frequency of infected CD4+ T cells and low CD4 counts on ART has been reported several times. However, these studies did not control for CD4 nadir or time on ART, so it is not clear whether SoIRs have a higher burden of infected CD4+ T cells. We hypothesize that increased induction from the HIV latent reservoir (LR), whether because of a larger LR size or increased inducibility from the LR, is correlated with suboptimal immune response. LR size and inducibility have never been simultaneously evaluated, but we will do so using efficient new assays that can discriminate intact from defective HIV proviruses. We will determine whether LR size and inducibility contribute to suboptimal immune response and whether cytokine therapies designed to increase CD4 counts also expand the LR. For Aim 1, we will determine whether the size of the HIV LR in blood and lymphoid tissue is positively correlated with suboptimal immune response using the new intact proviral DNA assay (IPDA), a droplet digital PCR assay that separately quantifies intact and defective proviruses, on samples from SoIRs and age- and nadir-matched controls identified from within the ACTG Longitudinal Linked Randomized Trials study and three large cohorts in Baltimore, San Francisco, and Cleveland. For Aim 2, we will determine whether infected CD4+ T cells of SoIRs are more readily inducible from latency using a quantitative viral induction assay on blood samples from SoIRs and matched controls. For Aim 3, we will determine whether cytokine therapies that increase CD4 count also expand the HIV LR by using the IPDA to measure LR size in samples from clinical trials of exogenous IL-7, IL-15, and IL-2 in treated HIV. Through formal didactic training and structured mentorship from experts in HIV reservoirs, HIV immunology, clinical research, and biostatistics, the PI will develop a unique skillset in HIV latency techniques, immunological techniques and knowledge, statistics, and translational research. This training provides a pathway to an independent career as a translational investigator researching the contribution of viral factors to the pathogenesis of treated HIV.